Scholars Academic Journal of Pharmacy (SAJP)
ISSN 2320-4206
Sch. Acad. J. Pharm., 2013; 2(2):41-46
©Scholars Academic and Scientific Publisher
(An International Publisher for Academic and Scientific Resources)
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Research Article
A GC-MS method for the Determination of Mancozeb and Metiram (as CS 2)
residues in Aquatic Tox medium
1
1
Tentu. Nageswara Rao, 3D. Sreenivasulu, 2T.B. Patrudu, 1K.M.S Sreenivas, 1Botsa. Parvatamma.
Department of Analytical Chemistry, International Institute of Bio-technology and Toxicology (IRO), Padappai,
Chennai- 601 301, Tamil Nadu, India.
2
Department of Engineering Chemistry, GITAM University, Hyderabad Campus.
3
Orchid Chemicals & Pharmaceuticals Ltd, Chennai.
*Corresponding author
1
Tentu. Nageswara Rao
Email: [email protected]
Abstract: A simple, and sensitive validated GCMS-EI analytical method was developed for the determination of
dithiocarbamates (Mancozeb and Metiram) residues in different aquatic tox mediums. The tox mediums were those
which provide nutrients and help the growth of different aquatic organisms for their survival and multiplication. The
constituent of different mediums includes blended water for fish, M4 Medium for Daphnia magna, OECD TG 201
medium for Alga and 20XAAP Medium for lemna. The
dithiocarbamates residue involves the reduction of
ditioncarbonate moiety under strong acidic conditions in presence of stannous chloride (SnCl2) as reducing agent,
evolution of carbon disulfide (CS2) insitu extraction of the CS2 into a layer of iso-octane subsequently analysis of
CS2 content by GC–MS in SIM mode. The established optimum conditions provided 5.0 min retention for CS2 and the
total time of chromatographic analysis was 7.0 min. The linear graph has the lowest detection 0.01 mg/L. The lowest
limit of quantification was 0.03 mg/L. The method was validated at levels of 0.03 and 0.3 mg/L. Recovery spiked
samples of mancozeb and Metiram in different mediums were in the range 91-98%, with relative standard deviations
0.78 to 2.2 % (n=5). The proposed method can be applied successfully for the determination of dithiocarbamate residues
in different aquatic solutions.
Keywords: Dithiocarbamates, Carbon disulfide, Aquatic Tox medium, GC-MS-EI method.
INTRODUCTION
The ditihocarbamate fungicides used in crop
protection services are the complex metal salts which
contains manganese (maneb), iron (ferbam), zinc
(zineb, mancozeb, propineb). The dithiocarbamates
posses the potential for the treatment of broad spectrum
of pathogens of mre than 400 species over 70 crops [1].
They were characterized based on their potential
activity against different plant pathogens. In
combination with modern systemic fungicide, they were
also used to manage resistance and to broaden the
spectrum of activity. The so called “maneb group”
(zineb, maneb, mancozeb, propineb, metiram)
fungicides are most frequently detected chemical in
several of the export commodities like grapes, and that
this group also had the highest frequency in exceeding
maximum residue limits (MRLs) [2]. The World Health
Organization (WHO)
classified few of the
dithiocarbamates as being hazardous [3] Which lead to
use of an array of different methods for the analysis of
their residues in different substrates.
The general methods of analysis are based on the
decomposition of dithiocarbamates to liberate carbon
disulfide (CS2) using lead acetate or by hot mineral acid
to the amines.
The liberated CS2 is subsequently
trapped in a digestion solvent and the active ingredient
is determined by Iodometric titrations [4-13]. Several of
these published methods are titrations methods and
suffers from practical difficulties while analyzing the
active by titration due to the interference associated
with the dirty samples. Some times the CS2 liberation
may not be complete or it may leak while trapping or
the reverse flow may contribute to the negative results
forcing the analyst to do multiple sample analysis.
EXPERIMENTAL PROCEDURES
Instrumentation
GC-MS conditions for the determination of
Dithiocarbamates
The configuration of GC–MS system used
includes a GC-17A (Shimadzu, Kyoto, Japan) gas
chromatograph coupled with QP-5050A Mass-Selective
Detection (MSD) and GC Solution software, the
detector was set in selective ion monitoring mode (SIM)
mode. The ions m/z 76 and m/z 78 were used as
qualifier ions (Figure 1) and the target ion used for the
measurement was the ion at m/z 76. The CS2 peak
separation was obtained on a Supelco SPB-1 capillary
column (30 m length, 0.32 mm internal diameter,
4.0µm film thickness). The injection system was
operated in split mode with a split ratio of 10:1. The
injector and the transfer line temperatures were 250ºC
and 300ºC, respectively. The oven temperature program
was 30ºC, held constant for 4.5 min and ramp at 70ºC
/min raised the column temperature up to 120ºC, held
41
Tentu. Nageswara Rao et al., Sch. Acad. J. Pharm., 2013; 2(2):41-46
constant for 3.0 min. The carrier gas used was helium
(purity 99.999%) at a flow rate of 2.0 ml /min and the
sample volume injected onto the column was 1.0 µL. A
Shimadzu GCMS solutions Chromatography Software
was used for acquisition of data and calculation of peak
area.. The carrier gas used was helium (purity 99.999%)
at a flow rate of 2.0 ml /min and the sample volume
injected onto the column was 1.0 µL. A Shimadzu
GCMS solution Chromatography Software was used for
acquisition of data and calculation of peak areas. The
retention time of CS2 was 5.0 min and the total time of
chromatographic analysis was 7.0 min.
Analytical standards, Reagents and Solutions
The analytical standard materials of Mancozeb
(purity 82.10% ), Metiram (purity 85.80% ) were
obtained from was purchased from sigma Aldrich. The
hydrochloric acid, ethylenediaminetetraacetic acid
disodium salt (EDTA) used were AR grade, methanol,
acetonitrile and Isooctane of HPLC grade were purchased
form Merck (Darmstadt, Germany). Stannous chloride
(purity 98%) was obtained from Aldrich. Milli-Q water
was obtained from Millipore India Ltd, Bangalore, India.
Analytical standard solutions of mancozeb and Metiram
were made in 0.2 M Disodium EDTA. Purity correction
and CS2 conversion factor was incorporated in the
preparation of analytical standard solutions. For each
pesticide a stock solution of 500 mg/L was prepared,
which was serially diluted to produce working standard
solutions. The working standard solutions were prepared
freshly and used.
Stock solutions of Na2EDTA (100g/L) and 8N HCl
were prepared in Milli-Q water. 3% Stannous chloride
solution was prepared in 8N hydrochloric acid.
Test medium
Test medium is a constitute of different macro
nutrients, salts and vitamins. This helps in the survival
of different organisms during exposure of different
compounds.
Blended water
It is a mixture of well water and reverse
osmosis water in the ratio of 1:1.7 liters. This provides
enough nutrients for the survival of fish during test item
exposure.
M4 Medium
It is a combination of Trace elements, Marco
nutrients and vitamins .The composition was given in
Table 1.
OECD TG 201 medium
This helps in the growth of green alga as it
provides the required nutrients and useful salts which
helps in their growth and multiplication.
The
composition was given in Table 2.
20X AAP Medium
This medium with different constitutes of
nutrients will help in the growth and survival of alga
during test item exposure.The composition details were
given in Table 3.
Digestion procedure
To a known volume of
homogenized
standard/sample taken in 250 ml crimp top vials, 5ml of
10% Na2 EDTA solution in boiled distilled water was
added, swirled the contents to mix, then 15 ml of
HCL/SnCl2 [8 N / 3%(w/v)] mixture was added along
with 20 ml of iso-octane and immediately capped the
crimp top vials. The vials were placed in a hot air oven
at 95° C for one hour. After digestion period the Isooctane layer was collected in GC vials and injected in to
GC-MS.
Method validation
The method for the determination of
dithiocarbamate residues was validated in terms of
method specificity, linearity, assay accuracy, precision,
limit of determination and quantification. The results
are expressed in term of its CS2 content.
Method specificity
Specificity was confirmed by injecting the isooctane trap of medium (Aqua Medium meant for fish
toxic study, M4 Medium, OECD TG 201 and 20X AAP
Medium).
Linearity
The calibration solution was prepared by digesting
analytical standards in stannous chloride solution and
trapped the liberated CS2 in iso-octane. The linearity of
the method was evaluated by preparing different
calibration solutions. A series of calibration solutions
were prepared by diluting the stock solution into 10 mL
volumetric flasks, and brought to volume with dimethyl
sulfoxide. The working calibration solutions were
pipetted into separate clean 250 mL capacity crimp top
vials giving rise to a series of solutions containing
0.01, 0.10, 1, 5, 10, 50 and 100 µg/mL. All the standard
solutions were processed and qualitative analysis was
based on the retention time (5.0 min) of carbon
disulfide by injecting carbon disulfide solutions made in
Iso-octane.
Assay accuracy and Repeatability
Recovery studies were carried out at 0.01 and
0.1 mg/L fortification levels for Mancozeb, and
Metiram (n=5 for each at two fortification levels) by
spiking 10 mL (aquatic mediums meant for fish,
daphnia, alga and lemma ) samples with the appropriate
volumes of working standard solutions. After spiking,
samples were handled and processed as above described
digestion procedure.
Detection and Quantification Limits
The lowest limit of detection (LOD, mg/L)
was determined as the lowest concentration giving a
42
Tentu. Nageswara Rao et al., Sch. Acad. J. Pharm., 2013; 2(2):41-46
response of 3 times the baseline noise defined from the
analysis of three control (untreated) samples. The limit
of quantification (LOQ, mg/L) was determined as the
lowest concentration of a given pesticide giving a
response of 10 times the baseline noise.
Stability of Analytical standard stock solutions
To determine the stability of the analytical
standard solutions, the calibration solutions were stored
under refrigerated conditions (4-8ºC) for a period of 30
days and aliquots was analyzed.
RESULTS
Specificity
There were no matrix peaks in the
chromatograms to interfere with the analysis of CS2
shown in Figure. 2. Furthermore, the retention time of
CS2 was relatively constant at 5.0 ± 0.2 min.
Linearity
Calibration curve at all concentration ranges
were better described by quadratic equation with
correlation coefficient > 0.99. Good linear correlation
coefficients (R2=0.999) between concentration (x) and
peak area (Y) were also found at lower concentration
ranges.
Assay accuracy and precision
The recovery data for various mediums are
shown in Table 1. Recoveries of mancozeb, and
Metiram were > 91%. The method was validated over
the fortification level 0.01 – 0.1 mg/L. The relative
standard deviation values obtained by this method are
summarized in Table 1. These numbers were calculated
from five (5) replicate analyses of a given sample
(mancozeb and Metiram) made by a single analyst on
one day. The repeatability of the method is satisfactory
(RSDs <5 % ).
Table 1: Preparation of M4 medium Nutrients (Daphnia Magna)
Sl.N
Chemical Name
Boric acid
Manganese chloride
Lithium chloride
Rubidium chloride
Strontium chloride
Sodium bromide
Sodium molybdate
Formula
o
.
Trace elements
H3BO3
MnCl2.4H2O
LiCl
RbCl
SrCl2.6H2O
NaBr
Na2MoO4.2H2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
mg/l
57190
7210
6120
1420
3040
320
1230
Cupric chloride
Zinc chloride
Cobalt chloride
Potassium iodide
Sodium selinite
Ammonium vanadate
EDTA*
CuCl2.2H2O
ZnCl2
CoCl2.6H2O
KI
Na2SeO3
NH4VO3
Na2EDTA.2H2
335
260
200
65
43.8
11.5
5000
O
O
FeSO4.7H2O
1991
Macro nutrients
16
Calcium chloride
CaCl2.H2O
293800
17
Magnesium sulphate
MgSO4.7H2O
246600
18
Potassium chloride
KCl
58000
19
Sodium hydrogen
NaHCO3
64800
20
Sodium silicate
Na2SiO3.9H2O
50000
21
Sodium nitrate
NaNO3
2740
22
Potassium phosphate
KH2PO4
1430
23
Potassium phosphate
KH2PO4
1840
Vitamin stock solutions
24
Thiamine hydrochloride
-------750
25
Cyanocobalamine (B12)
-------10
26
Biotin
-------7.5
* Both EDTA and Ferrous sulphate solution were prepared separately, poured together and autoclaved.
15
Ferrous sulphate*
43
Tentu. Nageswara Rao et al., Sch. Acad. J. Pharm., 2013; 2(2):41-46
TABLE 2: PREPARATION OF OECD TG 201 MEDIUM (GREEN ALGA)
Sl. No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Composition
NaHCO3 (Sodium Hydrogen Carbonate)
NH4Cl (Ammonium Chloride)
MgCl2.6H2O (Magnesium Chloride)
CaCl2.2H2O (Calcium Chloride)
MgSO4.7H2O (Magnesium Sulphate)
KH2PO4 (Potassium Dihydrogen Phosphate)
FeCl3.6H2O (Ferric Chloride)
Na2EDTA.2H2O (E.D.T.A. Disodium Salt)
H3BO3 (Boric Acid)
MnCl2.4H2O (Manganese (II) Chloride)
ZnCl2 (Zinc Chloride)
CoCl2.6H2O (Cobaltous Chloride)
Na2MoO4.2H2O (Sodium Molybdate)
CuCl2.2H2O (Copper (II) Chloride)
mg/L
50.0
15.0
12.0
18.0
15.0
1.60
0.064
0.100
0.185
0.415
0.0030
0.0015
0.0070
0.00001
TABLE 3: PREPARATION OF 20X AAP medium (LEMNA GIBBA)
Stock
Solution
No.
A1
A2
A3
B
C
Composition
NaNO3 (Sodium Nitrate)
MgCl2.6H2O (Magnesium Chloride)
CaCl2.2H2O (Calcium Chloride)
MgSO4.7H2O (Magnesium Sulphate)
K2HPO4.3H2O (Dipotassium Hydrogen Phosphate)
H3BO3 (Boric Acid)
MnCl2.4H2O (Manganese (II) Chloride)
FeCl3.6H2O (Ferric Chloride)
Na2EDTA.2H2O (E.D.T.A. Disodium Salt)
ZnCl2 (Zinc Chloride)
CoCl2.6H2O (Cobaltous Chloride)
Na2MoO4.2H2O (Sodium Molybdate)
CuCl2.2H2O (Copper (II) Chloride)
NaHCO3 (Sodium Hydrogen Carbonate)
Concentration
in stock
solution (g/L)
26.0
12.0
4.4
15.0
1.4
0.19
0.42
0.16
0.30
3.3 mg/L
1.4 mg/L
7.3 mg/L
0.012 mg/L
15
Concentration in
prepared
medium (mg/L)
510
240
90
290
30
3.7
8.3
3.2
6.0
66 µg/L
29 µg/L
145 µg/L
0.24 µg/L
300
Table 4: Recoveries of the three dithiocarbamates tested from fortified different mediums
Medium
Blended
water
OECD TG
201
M4
20XAAP
Fortification
level
(mg/L)
0.01
0.1
0.01
0.1
0.01
0.1
0.01
0.1
Mancozeb
Mean
Recovery
% RSD
(%)
98.12
2.36
97.56
2.02
96.58
1.58
95.87
2.14
96.75
1.45
97.95
1.69
97.65
1.84
95.99
2.78
Average of five replications
Metiram
Mean
Recovery
% RSD
(%)
98.25
2.73
98.42
0.98
96.40
1.64
95.76
1.21
100.18
2.32
98.62
1.28
97.45
1.75
98.24
2.03
44
Tentu. Nageswara Rao et al., Sch. Acad. J. Pharm., 2013; 2(2):41-46
Figure 1: Representative GC-MS scanned Chromatogram at m/z 76 and m/z 78 ions of standard dithiocarbamate
Figure 2: Representative GC-MS Chromatogram of standard dithiocarbamate tested from fortified medium
Figure 3: Representative GC-MS Chromatogram at fortification level of 0.01 mg/L
Detection and Quantification Limits
The limit of quantification was determined to
be 0.01 mg/L. this quantization limit was defined as the
lowest fortification level evaluated at which acceptable
average recoveries were 91-98%, RSD <3%. This
quantification limit also reflects the fortification level at
which an analyte peak is consistently generated at a
level approximately 10 times the baseline noise in the
chromatogram.The limit of detection was determined to
be 0.003 mg/L at a level of approximately three times
the back ground of control injection around the
retention time of the peak of interest. The LOD and
LOQ values were 0.003 mg/L and 0.01 mg/L.
Stability of analytical standard stock solutions
It is well-known that dithiocarbamates are not
stable in solution (30,31), From the storage stability
test (4-8ºC) it was concluded that mancozeb
decomposed at a rate of 4% per day, and only 20-25%
of the initial concentration was recovered after 1 week
of storage. On other hand, solution of Metiram was
stable, 84% and 87% of the initial concentrations were
recovered respectively after one week of storage.
Nevertheless, it is recommended that for all tested
compounds fresh stock and working standard solutions
are made for the daily update of calibration curves and
recovery studies.
45
Tentu. Nageswara Rao et al., Sch. Acad. J. Pharm., 2013; 2(2):41-46
Conclusion
Appropriate analytical methodology for the
determination of
dithiocarbamate( Mancozeb and
Metiram) residues in aquatic tox medium (meant for
fish, Daphnia, Algae and lemna) has been established
and validated. This method indirectly measures the
residual dithiocarbamates as concentration of Carbon
disulphide. This was found to be satisfactory in terms
of linearity of response, system precision, assay
accuracy and quantification.
Acknowledgement
The authors are thankful to the S. Sreenivas.,
Scientist. Krish biotech for his keen interest and help.
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